Work described herein was supported by funding from the National Institutes of Health. The United States Government has certain rights in the invention.
Pattern formation is the activity by which embryonic cells form ordered spatial arrangements of differentiated tissues. The physical complexity of higher organisms arises during embryogenesis through the interplay of cell-intrinsic lineage and cell-extrinsic signaling. Inductive interactions are essential to embryonic patterning in vertebrate development from the earliest establishment of the body plan, to the patterning of the organ systems, to the generation of diverse cell types during tissue differentiation (Davidson, E., (1990) Development 108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al., (1992) Cell 68: 257-270). The effects of developmental cell interactions are varied. Typically, responding cells are diverted from one route of cell differentiation to another by inducing cells that differ from both the uninduced and induced states of the responding cells (inductions). Sometimes cells induce their neighbors to differentiate like themselves (homoiogenetic induction); in other cases a cell inhibits its neighbors from differentiating like itself. Cell interactions in early development may be sequential, such that an initial induction between two cell types leads to a progressive amplification of diversity. Moreover, inductive interactions occur not only in embryos, but in adult cells as well, and can act to establish and maintain morphogenetic patterns as well as induce differentiation (J. B. Gurdon (1992) Cell 68:185-199).
Several classes of secreted polypeptides are known to mediate the cell-cell signaling that determines tissue fate during development. An important group of these signaling proteins are the TGFxcex2 superfamily of molecules, which have wide range of functions in many different species. Members of the family are initially synthesized as larger precursor molecules with an amino-terminal signal sequence and a pro-domain of varying size (Kingsley, D. M. (1994) Genes Dev. 8:133-146). The precursor is then cleaved to release a mature carboxy-terminal segment of 110-140 amino acids. The active signaling moiety is comprised of hetero- or homodimers of the carboxy-terminal segment (Massague, J. (1990) Annu. Rev. Cell Biol. 6:597-641). The active form of the molecule then interacts with its receptor, which for this family of molecules is composed of two distantly related transmembrane serine/threonine kinases called type I and type II receptors (Massague, J. et al. (1992) Cell 69:1067-1070; Miyazono, K. A. et al. EMBO J. 10:1091-1101). TGFxcex2 binds directly to the type II receptor, which then recruits the type I receptor and modifies it by phosphorylation. The type I receptor then transduces the signal to downstream components, which are as yet unidentified (Wrana et al, (1994) Nature 370:341-347).
Several members of the TGFxcex2 superfamily have been identified which play salient roles during vertebrate development. Dorsalin is expressed preferentially in the dorsal side of the developing chick neural tube (Basler et al. (1993) Cell 73:687-702). It promotes the outgrowth of neural crest cells and inhibits the formation of motor neuron cells in vitro, suggesting that it plays an important role in neural patterning along the dorsoventral axis. Certain of the bone morphogenetic proteins (BMPs) can induce the formation of ectopic bone and cartilage when implanted under the skin or into muscles (Wozney, J. M. et al. (1988) Science 242:1528-1534). In mice, mutations in BMP5 have been found to result in effects on many different skeletal elements, including reduced external ear size and decreased repair of bone fractures in adults (Kingsley (1994) Genes Dev. 8:133-146). Besides these effects on bone tissue, BMPs play other roles during normal development. For example, they are expressed in non skeletal tissues (Lyons et al. (1990) Development 109:833-844), and injections of BMP4 into developing Xenopus embryos promote the formation of ventral/posterior mesoderm (Dale et al (1992) Development 115:573-585). Furthermore, mice with mutations in BMP5 have an increased frequency of different soft tissue abnormalities in addition to the skeletal abnormalities described above (Green, M. C. (1958) J. Exp. Zool. 137:75-88).
Members of the activin subfamily have been found to be important in mesoderm induction during Xenopus development (Green and Smith (1990) Nature 47:391-394; Thomsen et al. (1990) Cell 63:485-493) and inhibins were initially described as gonadal inhibitors of follicle-stimulating hormone from pituitary cells. In addition, antagonists of this signaling pathway can be used to convert embryonic tissue into ectoderm, the default pathway of development in the absence of TGFxcex2-mediated signals. BMP-4 and activin have been found to be potent inhibitors of neuralization (Wilson, P. A. and Hemmati-Brivanlou, A (1995) Nature 376:331-333).
Further evidence for the importance of a TGFxcex2 family member in early vertebrate development comes from a retroviral insertion in the mouse nodal gene. This insertion leads to a failure to form the primitive streak in early embryogenesis, a lack of axial mesoderm tissue, and an overproduction of ectoderm and extraembryonic ectoderm (Conlon et al. (1991) Development 111:969-981; lannaccone et al (1992) Dev. Dynamics 194:198-208). The predicted nodal gene product is consistent with previous studies showing that nodal is related to activins and BMPs (Zhou et al. (1993) Nature 361:543-547). A role for TGFxcex2 family members in the development of sex organs has also been described; Mullerian inhibitory substance functions during vertebrate male sexual development to cause regression of the embryonic duct system that develops into oviducts and uterus (Lee and Donahoe (1993) Endocrinol. Rev. 14:152-164).
Members of this family of signaling molecules also continue to function post-development. TGFxcex2 has antiproliferative effects on many cell types including epithelial cells, endothelial cells, smooth muscle cells, fetal hepatocytes, and myeloid, erythroid, and lymphoid cells. Animals which cannot produce TGFxcex21 (homozygous for null mutations in the TGFxcex21 gene) have been found to survive until birth with no apparent morphological abnormalities (Shull et al. (1992) Nature 359:693-699; Kulkarni et al. (1993) Proc. Natl. Acac. Sci. 90:770-774). The animals do die around weaning age, however, owing to massive immune infiltration in may different organs. These data are consistent with the inhibitory effects of TGFxcex2 on lymphocyte growth (Tada et al. (1991) J. Immunol 146:1077-1082). In another system, the expression of a TGFxcex2 transgene in the mammary tissue of mice has been shown to inhibit the development and secretory function of mammary tissue during sexual maturation and pregnancy (Jhappan, C. et al. (1993) EMBO J. 12:1835-1845; Pierce, D. F. et al. (1993) Genes Dev. 7:2308-2317). In addition to these inhibitory effects, TGFxcex2 can also promote the growth of other cell types as evidenced by its role in neovascularization and the proliferation of connective tissue cells. Because of these activities, it plays a key role in wound healing (Kovacs, E. J. (1991) Immunol Today 12:17-23)
The present invention relates to the discovery of a novel family of genes, and gene products, expressed in vertebrate organisms, which genes referred to hereinafter as the xe2x80x9csignalinxe2x80x9d gene family, the products of which are referred to as signalin proteins. The products of the signalin gene have apparent broad involvement in the formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, and can be used or manipulated to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.
In general, the invention features isolated vertebrate signalin polypeptides, preferably substantially pure preparations of one or more of the subject signalin polypeptides. The invention also provides recombinantly produced signalin polypeptides. In preferred embodiments the polypeptide has a biological activity including: an ability to modulate proliferation, survival and/or differentiation of mesodermally-derived tissue, such as tissue derived from dorsal mesoderm; the ability to modulate proliferation, survival and/or differentiation of ectodermally-derived tissue, such as tissue derived from the neural tube, neural crest, or head mesenchyme; the ability to modulate proliferation, survival and/or differentiation of endodermally-derived tissue, such as tissue derived from the primitive gut. Moreover, in preferred embodiments, the subject signalin proteins have the ability to modulate intracellular signal transduction pathways mediated by receptors for members of the Transforming Growth Factor xcex2 superfamily of molecules.
In one embodiment, the polypeptide is identical with or homologous to a signalin protein. Exemplary signalin proteins are represented by SEQ ID No. 14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No: 20, SEQ ID No:21, SEQ ID No:22, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, SEQ ID No:26. Related members of the vertebrate signalin family are also contemplated, for instance, a signalin polypeptide preferably has an amino acid sequence at least 60% homologous to a polypeptide represented by any of SEQ ID Nos: 14-26, though polypeptides with higher sequence homologies of, for example, 70, 80%, 90% or are also contemplated. The signalin polypeptide can comprise a full length protein, such as represented in the sequence listings, or it can comprise a fragment corresponding to particular motifs/domians, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150 or 200 amino acids in length. In preferred embidiments, the polypeptide, or fragment thereof, specifically modulates, by acting as either an agonist or antagonist, the signal transduction activity of a receptor for a transforming growth factor xcex2.
In certain preferred embodiments, the invention features a purified or recombinant signalin polypeptide having a molecular weight in the range of 45 kd to 70 kd. For instance, preferred signalin polypeptide chains of the xcex1 and xcex2 subfamilies, described infra, have molecular weights in the range of 45 kd to about 55 kd, even more preferably in the range of 50-55 kd. In another illustrative example, preferred signalin polypeptide chains of the xcex3 subfamily have molecular weights in the range of 60 kd to about 70 kd, even more preferably in the range of 63-68 kd. It will be understood that certain post-translational modifications, e.g., phosphorylation and the like, can increase the apparent molecular weight of the signalin protein relative to the unmodified polypeptide chain.
In another embodiment, the signalin polypeptide comprises a signalin motif represented in the general formula shown in SEQ ID No:28. In a preferred embodiment the signalin motif corresponds to a signalin motif represented in one of SEQ ID Nos: 14-26. In another embodiment, the signalin polypeptide of the invention comprises a xcexd domain represented in the general formula SEQ ID No:27. In a preferred embodiment the xcexd region corresponds to a xcexd domain represented in one of SEQ ID Nos:14-26. In another preferred embodiment, the signalin polypeptide of the invention comprises a "khgr" domain represented in the general formula SEQ ID No:29. In a further preferred embodiment the "khgr" region corresponds to a "khgr" domain represented in one of SEQ ID Nos:14-26. In another perferred embodiment, the signalin polypeptide can modulate, either stimulate or antagonize, intracellular pathways mediated by a receptor for a TGFxcex2. In still another embodiment, the polypeptide comprises an amino acid sequence represented in the general formula: LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXECCEXPFXSKQKXV (SEQ ID No:30). In still a further embodiement, the signalin polypeptide of the present invention comprises an amino acid sequence represented by the general formula: LDGRLQVAGRKGFPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV (SEQ ID No:31). In an additional embodiement, the signalin polypeptide of the present invention comprises an amino acid sequence represented by the general formula: LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEV (SEQ ID No:32).
In another preferred embodiment, the invention features a purified or recombinant polypeptide fragment of a signalin protein, which polypeptide has the ability to modulate, e.g., mimic or antagonize, a the activity of a wild-type signalin protein. Preferably, the polypeptide fragment comprises a signalin motif.
Moreover, as described below, the preferred signalin polypeptide can be either an agonist (e.g. mimics), or alternatively, an antagonist of a biological activity of a naturally occurring form of the protein, e.g., the polypeptide is able to modulate differentiation and/or growth and/or survival of a cell responsive to authentic signalin proteins. Homologs of the subject signalin proteins include versions of the protein which are resistant to post-translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which inactivate an enzymatic activity associated with the protein.
The subject proteins can also be provided as chimeric molecules, such as in the form of fusion proteins. For instance, the signalin protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the signalin polypeptide, e.g. the second polypeptide portion is glutathione-S-transferase, e.g. the second polypeptide portion is an enzymatic activity such as alkaline phosphatase, e.g. the second polypeptide portion is an epitope tag.
In a preferred embodiment the signalin polypeptide of the present invention modulates signal transduction from a TGFxcex2 receptor. For example, the signalin polypeptide may modulate the transduction of a TGFxcex2 receptor for a member of the dpp family, e.g., dpp, BMP2, or BMP4. In a other preferred embodiement, the signalin polypeptide modulates the signaling of a TGFxcex2 other than a dpp family member. For instance, the signalin polypeptide may be involved in signalling from one or more of BMP5, BMP6, BMP7, BMP8, 60A, GDF5, GDF6, GDF7, GDF1, Vg1, dorsalin, BMP3, GDF10, nodal, inhibins, activins TGFxcex21, TGFxcex22, TGFxcex23, MIS, GDF9 or GDNE.
In yet another embodiment, the invention features a nucleic acid encoding a signalin polypeptide, or polypeptide homologous thereto, which polypeptide has the ability to modulate, e.g., either mimic or antagonize, at least a portion of the activity of a wild-type signalin polypeptide. Exemplary signalin polypeptides are represented by SEQ ID No:14, SEQ ID No:15, SEQ ID No. 16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, SEQ ID No: 22, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, SEQ ID No:26. In another embodiment the nucleic acid of the present invention hybridizes under stringent conditions with one or more of the nucleic acid sequences in SEQ ID No:1-13. In preferred embidiments, the nucleic acid encodes a polypeptide which specifically modulates, by acting as either an agonist or antagonist, the signal transduction activity of a receptor for a transforming growth factor xcex2.
In another embodiment, the nucleic acid encodes an amino acid sequence which comprises a signalin motif represented in the general formula shown in SEQ ID No:28. In preferred embodiment the signalin motif corresponds to a signalin motif represented in one of SEQ ID Nos:14-26. In another embodiment, the nucleic acid of the invention encodes an amino acid sequence which comprises a xcexd domain represented in the general formula SEQ ID No:27. In a preferred embodiment the encoded xcexd region corresponds to a xcexd domain represented in one of SEQ ID Nos:14-26. In another embodiment, the nucleic acid encodes a signalin polypeptide of the invention which comprises a "khgr" domain represented in the general formula SEQ ID No:29. In a preferred embodiment the encoded "khgr" region corresponds to a "khgr" domain represented in one of SEQ ID Nos:14-26. In still a another embodiment, the nucleic acid sequence encodes a polypeptide which comprises an amino acid sequence represented in the general formula: LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXECCEXPFXSKQKXV (SEQ ID NO: 30). In another embodiement, the nucleic acid of the present invention encodes a polypeptide which comprises an amino acid sequence represented by the general formula, LDGRLQVAGRKGFPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV (SEQ ID NO: 30). In an still another embodiement, the nucleic acid encodes a polypeptide which comprises an amino acid sequence represented by the general formula, LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEV (SEQ ID NO. 32).
Another aspect of the present invention provides an isolated nucleic acid having a nucleotide sequence which encodes a signalin polypeptide. In preferred embodiments, the encoded polypeptide specifically mimics or antagonizes inductive events mediated by wild-type signalin proteins. The coding sequence of the nucleic acid can comprise a sequence which is identical to a coding sequence represented in one of SEQ ID Nos: 1-13, or it can merely be homologous to one or more of those sequences.
Furthermore, in certain preferred embodiments, the subject signalin nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the signalin gene sequence. Such regulatory sequences can be used in to render the signalin gene sequence suitable for use as an expression vector. This invention also contemplates the cells transfected with said expression vector whether prokaryotic or eukaryotic and a method for producing signalin proteins by employing said expression vectors.
In yet another embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of either sense or antisense sequence of one or more of SEQ ID Nos:1-13; though preferably to at least 25 consecutive nucleotides; and more preferably to at least 40, 50 or 75 consecutive nucleotides of either sense or antisense sequence of one or more of SEQ ID Nos:1-13.
Yet another aspect of the present invention concerns an immunogen comprising a signalin polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a signalin polypeptide, e.g. a humoral response, e.g. an antibody response; e.g. a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g. a unique determinant, from a protein represented by one of SEQ ID Nos. 14-26.
A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the signalin immunogen.
The invention also features transgenic non-human animals, e.g. mice, rats, rabbits, chickens, frogs or pigs, having a transgene, e.g., animals which include (and preferably express) a heterologous form of a signalin gene described herein, or which misexpress an endogenous signalin gene, e.g., an animal in which expression of one or more of the subject signalin proteins is disrupted. Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed signalin alleles or for use in drug screening.
The invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 12 consecutive nucleotides of sense or antisense sequence of SEQ ID No:1-13, or naturally occurring mutants thereof. Nucleic acid probes which are specific for each of the classes of vertebrate signalin proteins are contemplated by the present invention, e.g. probes which can discern between nucleic acid encoding an xcex1, xcex2, or xcex3 signalin. In preferred embodiments, the probe/primer further includes a label group attached thereto and able to be detected. The label group can be selected, e.g., from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Probes of the invention can be used as a part of a diagnostic test kit for identifying dysfunctions associated with mis-expression of a signalin protein, such as for detecting in a sample of cells isolated from a patient, a level of a nucleic acid encoding a subject signalin protein; e.g. measuring a signalin mRNA level in a cell, or determining whether a genomic signalin gene has been mutated or deleted. These so called xe2x80x9cprobes/primersxe2x80x9d of the invention can also be used as a part of xe2x80x9cantisensexe2x80x9d therapy which refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more of the subject signalin proteins so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation. Preferably, the oligonucleotide is at least 12 nucleotides in length, though primers of 25, 40, 50, or 75 nucleotides in length are also contemplated.
In yet another aspect, the invention provides an assay for screening test compounds for inhibitors, or alternatively, potentiators, of an interaction between a signalin protein and a signalin binding protein or nucleic acid sequence. An exemplary method includes the steps of (i) combining a signalin polypeptide or fragment thereof, a signalin binding element, and a test compound, e.g., under conditions wherein, but for the test compound, the signalin protein and binding element are able to interact; and (ii) detecting the formation of a complex which includes the signalin protein and the binding element either by directly quantitating the complex or by measuring inductive effects of the signalin protein. A statistically significant change, such as a decrease, in the formation of the complex in the presence of a test compound (relative to what is seen in the absence of the test compound) is indicative of a modulation, e.g., inhibition, of the interaction between the signalin protein and its binding element.
Yet another aspect of the present invention concerns a method for modulating one or more of growth, differentiation, or survival of a mammalian cell responsive to signalin induction. In general, whether carries out in vivo, in vitro, or in situ, the method comprises treating the cell with an effective amount of a signalin polypeptide so as to alter, relative to the cell in the absence of signalin treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell. Accordingly, the method can be carried out with polypeptides mimics the effects of a naturally-occurring signalin protein on the cell, as well as with polypeptides which antagonize the effects of a naturally-occurring signalin protein on said cell. In preferred embodiments, the signalin polypeptide provided in the subject method are derived from vertebrate sources, e.g., are vertebrate signalin polypeptides. For instance, preferred polypeptides includes an amino acid sequence identical or homologous to an amino acid sequence (e.g., including bioactive fragments) designated in one of SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, or SEQ ID No:12, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, SEQ ID No:26. Furthermore, the present invention contemplates the use of other metazoan (e.g., invertebrate) homologs of the signalin polypeptides or bioactive fragments thereof equivalent to the subject vertebrate fragments.
In one embodiment, the subject method includes the treatment of testicular cells, so as modulate spermatogenesis. In another embodiment, the subject method is used to modulate osteogenesis, comprising the treatment of osteogenic cells with a signalin polypeptide. Liekwise, where the treated cell is a chondrogenic cell, the present method is used to modulate chondrogenesis. In still another embodiment, signalin polypeptides can be used to modulate the differentiation of neural cells, e.g., the method can be used to cause differentiation of a neuronal cell, to maintain a neuronal cell in a differentiated state, and/or to enhance the survival of a neuronal cell, e.g., to prevent apoptosis or other forms of cell death. For instance, the present method can be used to affect the differentiation of such neuronal cells as motor neurons, cholinergic neurons, dopanergic neurons, serotenergic neurons, and peptidergic neurons.
The present method is applicable, for example, to cell culture technique, such as in the culturing of neural and other cells whose survival or differentiative state is dependent on signalin function. Moreover, signalin agonists and antagonists can be used for therapeutic intervention, such as to enhance survival and maintenance of neurons and other neural cells in both the central nervous system and the peripheral nervous system, as well as to influence other vertebrate organogenic pathways, such as other ectodermal patterning, as well as certain mesodermal and endodermal differentiation processes. In an exemplary embodiment, the method is practiced for modulating, in an animal, cell growth, cell differentiation or cell survival, and comprises administering a therapeutically effective amount of a signalin polypeptide to alter, relative the absence of signalin treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of one or more cell-types in the animal.
Another aspect of the present invention provides a method of determining if a subject, e.g. a human patient, is at risk for a disorder characterized by unwanted cell proliferation or aberrant control of differentiation. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding a signalin protein, e.g. represented in one of SEQ ID Nos: 14-26, or a homolog thereof; or (ii) the mis-expression of a signalin gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from a signalin gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of the protein.
For example, detecting the genetic lesion can include (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of a signalin gene, e.g. a nucleic acid represented in one of SEQ ID Nos: 1-13, or naturally occurring mutants thereof, or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the signalin gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the signalin gene and, optionally, of the flanking nucleic acid sequences. For instance, the probe/primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of a signalin protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the signalin protein.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.